Full length, wild-type human epidermal growth factor (EGF; see SEQ ID NO: 1) is a 53 amino acid protein with a molecular weight of 6217 daltons and a variety of biological functions (Karnes, W., Epidermal growth factor and transforming growth factor alpha, 1994, Raven Press, New York). Modifications of the amino acid sequence at the C-terminus have been reporting both from construction of altered forms by recombinant DNA engineering genetic studies, and as observed with EGF isolated from nature. EGF is susceptible to both endo and exo proteases, and proteolytic attack occurs in the stomach on EGF produced in the salivary gland and swallowed, has been observed as well as with EGF in the blood stream (Araki et al., Chem. Pharm. Bull. 37(2), 404-406, 1989; Playford et al., Gastronenterology, 108, 92-101, 1996).
EGF that is equal to or less than 46 amino acids in length has lost substantial biological activities, in comparison to EGF species of chain lengths equal to or greater than 47 amino acids. There are contradictory data on the exact effect on chain length on functional biological activities such as affinity for receptors and in vivo rates of EGF clearance. EGF of lengths 47, 48 and 51 amino acids (indicated “EGF47”, “EGF48”, etc.) can inhibit stomach acid secretion (U.S. Pat. No. 3,917,824). Human EGF47 inhibits acid secretion with the same potency as EGF53, however retains only about one tenth of the potency to stimulate fibroblast growth (mitogenic activity; Hollenberg et al., Molecular Pharmacology 17, 314-320, 1980; Gregory et al., Regulatory Peptides 22, 217-226, 1988). As these foregoing references show, the fact that a composition demonstrates high biological activity with respect to one biological activity does not imply that all biological activities are present in amounts equipotent to the full length composition.
EGF52 is equipotent to EGF53, in terms of both inhibition of acid secretion and stimulation of cell proliferation. The mitogenic activity of mouse EGF is largely lost if the chain length is less than 48 amino acids (Burgess et al., Biochemistry 27, 4977-4985, 1988). Further, hEGF51 and EGF53 display similar pharmacokinetics (Kuo et al., Drug Metabolism and Disposition 20, 23-30, 1992). EGF51 has similar activities as EGF53 (Calnan et al., Gut 47, 622-627, 2000), except for the retention of immunosuppressive activity (Koch et al., J. Molecular Biochemistry 25, 45-59, 1984). EGF48 is reported to be stable to proteolysis and to retain biological activities (U.S. Pat. No. 5,434,135; Kuo et al. op.cit; Sizemore et al. Peptides 17, 1229-1236, 1996). However, EGF48 has significantly lower activity than EGF 53 (Goodlad et al., Clinical Science, 91, 503-507, 1996).
Correct formation of disulfide bonds and its biological activity are not affected by shortening of the N-terminus by up to 5 amino acids (Shin, S. et al., Peptides 16, 205-210, 1995; DiAugustine et al., Analytical Biochemistry 165, 420-429, 1987). Oxidation of the methionine residue at position 21 does not affect the biological activity of recombinant h-EGF produced by yeast (George-Nascimento et al. Biochemistry, 27, 797-802).
Recombinant EGF is degraded by microbial proteases during production. Recombinant hEGF53 produced in Saccharomyces cerevisiae is degraded to a sequence of 52 and then to 51 amino acids in length, as a result of protease activity during fermentation (George-Nascimento, Biochemistry, 27, 797-802, 1988). EGF produced by Pichia pastoris is degraded to a form having 48 amino acids that is stable, and which is described as retaining high biological activity. (U.S. Pat. No. 5,102,789). Similarly, mouse EGF produced and secreted by Pichia pastoris is partly degraded during fermentation to 51 amino acids in length (Clare et al., Gene 105, 205-212, 1991).
Comparison of these data raises questions regarding susceptibilities to proteolysis of various forms of EGF, and correlations between the lengths of these different forms and the extent of several different biological activities. Because biological activities are mediated by a family of receptors that can be differently expressed, i.e., are tissue specific, any relevant biological activity of a modified EGF must be tested for that activity, to ascertain its level of function, since a biological activity level is not necessarily predictable from data obtained using an assay of another biological activity.
EGF and EGF receptor ligands such as TGFα have been shown to comprise a treatment for diabetes (Nardi et al., U.S. Pat. No. 5,885,956, issued Mar. 23, 1999; and Nardi et al., U.S. Pat. No. 6,288,301, issued Sep. 11, 2001), a disease that has achieved epidemic proportions in the United States and elsewhere.
There is a need for EGF proteins that are stable to proteolysis, that are produced as a single molecular species in high yields in a safe and convenient production organism with reproducible composition and purity required for approval as a drug, and that retain substantial biological activity for such a therapeutic purpose.